Recombinant Phenylobacterium zucineum Lipoyl synthase (lipA)

What is Lipoyl Synthase (LipA) and what is its function in cellular metabolism?

Lipoyl synthase (LipA) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the insertion of two sulfur atoms into the C-6 and C-8 positions of an octanoyl moiety attached to specific proteins, producing lipoic acid. Lipoic acid is an essential cofactor for the glycine cleavage system (GCS) involved in C1 compound metabolism and for 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids . This cofactor is found in all domains of life and is crucial for energy metabolism, allowing cells to convert energy from food into forms usable by cells . In bacteria like P. zucineum, LipA plays a vital role in central metabolism by enabling these key enzymatic complexes to function properly.

How does the structure of LipA enable its catalytic function?

LipA is classified as a member of the radical SAM superfamily, possessing the conserved sequence motif CX3CX2C that coordinates an iron-sulfur cluster . Most LipA enzymes, including those characterized from model organisms, contain two [4Fe-4S] clusters:

• A basic cluster that generates the deoxyadenosyl radical by cleaving S-adenosylmethionine (SAM)

• An auxiliary cluster that provides the sulfur atoms for insertion into the octanoyl substrate

The deoxyadenosyl radical initiates the reaction by abstracting a hydrogen atom from the C-6 carbon of the octanoyl group. The resulting carbon radical then reacts with a sulfur atom from the auxiliary [4Fe-4S] cluster, forming a C-S bond . This process occurs at both the C-6 and C-8 positions, followed by the addition of two protons to generate the reduced form of the lipoyl group . The structural arrangement of these clusters and the positioning of the substrate binding pocket are critical for the enzyme's function.

What are the typical expression systems used for producing recombinant P. zucineum LipA?

While specific expression systems for P. zucineum LipA are not directly mentioned in the search results, recombinant lipoyl synthases are typically expressed in E. coli systems using vectors that allow for inducible expression, such as pET vectors. Based on protocols used for other LipA enzymes, the following approach can be applied:

• Gene cloning: The lipA gene from P. zucineum can be PCR-amplified and cloned into an expression vector with an appropriate affinity tag (His-tag, for example)

• Expression conditions: Transformation into an E. coli strain like BL21(DE3) followed by induction with IPTG

• Anaerobic conditions: Since LipA contains oxygen-sensitive iron-sulfur clusters, expression and purification are often performed under anaerobic conditions

• Reconstitution: Following purification, the enzyme typically requires reconstitution of its iron-sulfur clusters in vitro to ensure full activity

This approach allows for the production of sufficient quantities of recombinant enzyme for biochemical and structural studies.

How can the iron-sulfur clusters of recombinant P. zucineum LipA be effectively reconstituted for maximal enzymatic activity?

Reconstitution of iron-sulfur clusters is critical for obtaining catalytically active LipA. Based on studies with other lipoyl synthases, the following methodology can be applied:

Protocol for Iron-Sulfur Cluster Reconstitution:

• Anaerobic environment preparation:

  • Perform all steps in an anaerobic chamber with O₂ levels below 1 ppm

  • Pre-degas all buffers by sparging with nitrogen or argon

• Reduction step:

  • Treat purified LipA with a reducing agent (typically 5-10 mM DTT) for 30 minutes at 4°C

• Cluster assembly:

  • Add ferric chloride (FeCl₃) or ferrous ammonium sulfate to a final concentration of 8-fold molar excess relative to protein

  • Add sodium sulfide (Na₂S) to the same concentration

  • Incubate for 2-4 hours at 4°C with gentle stirring

• Removal of excess reconstitution components:

  • Pass the reconstituted protein through a desalting column equilibrated with anaerobic buffer

• Activity verification:

  • Measure iron and sulfide content spectrophotometrically

  • Confirm [4Fe-4S] cluster formation by UV-visible spectroscopy (characteristic absorbance at ~410 nm)

  • Perform electron paramagnetic resonance (EPR) spectroscopy to verify cluster integrity

Research has demonstrated that proper reconstitution is essential for LipA activity. In studies with TK2109 and TK2248 proteins from T. kodakarensis, reactions with non-reconstituted proteins showed very low levels of product formation compared to the reconstituted enzymes . This indicates that the procedure to reconstitute [4Fe-4S] clusters is critical for generating an active lipoyl synthase enzyme.

What are the most reliable assays for measuring P. zucineum LipA enzymatic activity?

Several complementary approaches can be used to assess LipA activity:

HPLC-Based Activity Assay:

• Substrate preparation:

  • Synthesize an octanoyl-peptide substrate corresponding to the lipoyl domain of the target protein

  • For P. zucineum, this would typically be derived from its GCS H-protein or E2 subunit sequence

• Reaction setup:

  • Combine reconstituted LipA with:

    • Octanoyl-peptide substrate (50-200 μM)

    • S-adenosylmethionine (SAM) (0.5-2 mM)

    • Sodium dithionite or other electron donor (1-5 mM)

    • Buffer containing dithiothreitol (DTT)

• Analysis:

  • Separate reaction products by HPLC using a C18 reversed-phase column

  • Monitor at 220 nm (peptide bond absorption) and 333 nm (lipoyl group)

  • Compare retention times with standards (octanoyl-peptide and lipoyl-peptide)

LC-MS Confirmation:

• Analyze reaction products by liquid chromatography-mass spectrometry

• Key masses to monitor:

  • Octanoyl-peptide: [M+H]⁺ will depend on the specific peptide sequence

  • Intermediate thiol-octanoyl-peptide: [M+H]⁺ = original octanoyl-peptide mass + 32 Da (one sulfur)

  • Lipoyl-peptide (oxidized form): [M+H]⁺ = original octanoyl-peptide mass + 64 Da (two sulfurs)

  • Reduced lipoyl-peptide: [M+H]⁺ = oxidized form + 2 Da

This combination of techniques allows for robust confirmation of LipA activity and can also provide insights into reaction intermediates.

How does the mechanism of P. zucineum LipA differ from other characterized lipoyl synthases?

While specific information on P. zucineum LipA is not provided in the search results, comparing its mechanism to well-characterized LipA enzymes can reveal important functional differences:

Comparison of Potential Mechanisms:

Research on M. tuberculosis LipA has shown that the iron-sulfur cluster destroyed during lipoic acid production is replaced by an iron-sulfur carrier protein, NfuA, allowing LipA to continue producing lipoic acid . This represents a unique regeneration mechanism that might be conserved in P. zucineum LipA as well.

P. zucineum LipA likely follows the classical mechanism, but sequence analysis and biochemical characterization would be needed to confirm and identify any unique features of this enzyme.

What are the optimal conditions for expressing and purifying recombinant P. zucineum LipA?

Based on research with other lipoyl synthases, the following conditions would likely be optimal:

Expression Optimization:

• Host strain selection:

  • Use E. coli strains designed for iron-sulfur protein expression (e.g., BL21(DE3) supplemented with iron)

  • Consider co-expression with iron-sulfur cluster biogenesis genes (isc or suf operons)

• Growth conditions:

  • Medium: LB or M9 minimal medium supplemented with iron (50-100 μM ferric citrate)

  • Temperature: Lower temperature (16-18°C) after induction to enhance proper folding

  • Induction: Low IPTG concentration (0.1-0.2 mM) to prevent inclusion body formation

  • Duration: Extended expression (16-24 hours) at lower temperature

• Anaerobic considerations:

  • For maximal retention of iron-sulfur clusters, shift cultures to anaerobic conditions after induction

  • Supplement media with cysteine and ferrous iron under anaerobic conditions

Purification Strategy:

• Buffer composition:

  • Base buffer: 50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • Salt: 150-300 mM NaCl

  • Reducing agent: 5-10 mM DTT or 2 mM β-mercaptoethanol

  • Glycerol: 10% to enhance stability

• Purification steps:

  • Affinity chromatography (Ni-NTA for His-tagged protein)

  • Size exclusion chromatography to remove aggregates

  • All steps performed anaerobically to preserve iron-sulfur clusters

• Storage conditions:

  • Store in buffer containing 10-20% glycerol at -80°C

  • Flash-freeze small aliquots in liquid nitrogen

  • Maintain anaerobic conditions during storage

This approach minimizes oxygen exposure and maximizes the retention of iron-sulfur clusters, which are essential for enzymatic activity.

How can researchers troubleshoot issues with low activity of recombinant P. zucineum LipA?

When facing low activity of recombinant LipA, researchers should consider the following troubleshooting approach:

Systematic Troubleshooting Strategy:

• Verify protein integrity:

  • SDS-PAGE to confirm expected molecular weight

  • Western blot with anti-His or anti-LipA antibodies

  • Mass spectrometry to confirm full-length protein

• Assess iron-sulfur cluster content:

  • Measure iron and sulfide content (target: 8 Fe and 8 S per protein)

  • UV-visible spectroscopy to verify characteristic absorption at ~410 nm

  • EPR spectroscopy to assess cluster integrity

  • Color check: properly reconstituted LipA should be brownish

• Evaluate reconstitution efficiency:

  • If iron-sulfur content is low, repeat reconstitution

  • Try alternative reconstitution methods (chemical vs. enzymatic)

  • Co-express with iron-sulfur cluster assembly proteins

• Optimize assay conditions:

  • Ensure strictly anaerobic conditions during activity assays

  • Test different electron donors (sodium dithionite, flavodoxin/flavodoxin reductase)

  • Vary SAM concentration (0.25-2 mM)

  • Test different pH values (7.0-8.5)

  • Add cluster-stabilizing agents (DTT, glutathione)

• Address potential inhibition:

  • Check for inhibitory compounds in the buffer

  • Ensure SAM quality (commercial SAM can degrade)

  • Test for product inhibition

• Consider co-factors or partner proteins:

  • Based on findings with T. kodakarensis LipS1/LipS2, LipA might require a partner protein

  • Research from Penn State shows LipA requires NfuA to replace destroyed iron-sulfur clusters

  • Test adding potential carrier proteins to reaction mixture

Research has shown that even when individual components (like TK2109 or TK2248 proteins from T. kodakarensis) show no activity alone, they may function together as a lipoyl synthase . This highlights the importance of considering protein partnerships in LipA function.

What impact does the iron-sulfur cluster carrier protein have on P. zucineum LipA activity and how can this be studied?

The interaction between LipA and iron-sulfur cluster carrier proteins represents an important area for investigation:

Role and Impact of Carrier Proteins:

Research from Penn State University has demonstrated that an iron-sulfur cluster carrier called NfuA replaces the destroyed iron-sulfur cluster in LipA, allowing it to continue producing lipoic acid . Without this regeneration mechanism, LipA would be a single-turnover enzyme, severely limiting its biological utility.

Methodological Approaches to Study This Interaction:

• Co-expression studies:

  • Clone and co-express P. zucineum LipA with potential carrier proteins (NfuA, IscA, SufA)

  • Compare activity of LipA expressed alone versus co-expressed with carriers

  • Measure multiple turnover capability with and without carriers

• Protein-protein interaction analysis:

  • Pull-down assays using tagged LipA to identify interacting partners

  • Surface plasmon resonance to determine binding kinetics

  • Crosslinking studies followed by mass spectrometry

  • Yeast two-hybrid screening to identify novel interaction partners

• In vitro reconstitution experiments:

  • Design a multiple-turnover assay with:

    • Purified LipA

    • Octanoyl-peptide substrate in excess

    • SAM in excess

    • Electron donor system

    • ± purified carrier protein (NfuA or homologs)

  • Monitor reaction progress over time to detect cessation of activity

  • Add carrier protein at different timepoints to observe rescue of activity

• Structural studies:

  • Co-crystallize LipA with carrier protein

  • Cryo-EM analysis of the complex

  • HDX-MS (hydrogen-deuterium exchange mass spectrometry) to map interaction surfaces

Experimental Design for Testing Multiple Turnover:

Sample at multiple timepoints (0, 15, 30, 60, 120 min) and analyze product formation by HPLC or LC-MS. In the absence of carrier protein, product formation should plateau after a few turnovers as the auxiliary cluster is destroyed. With carrier protein present, sustained product formation would indicate successful cluster replacement and enzyme regeneration .

How do mutations in conserved cysteine motifs affect P. zucineum LipA activity and structural integrity?

Cysteine residues in LipA play crucial roles in coordinating iron-sulfur clusters essential for catalysis. Based on information from other lipoyl synthases, we can predict how mutations would affect activity:

Impact of Mutations in Key Motifs:

Methodology to Study Mutational Effects:

• Site-directed mutagenesis:

  • Generate cysteine-to-alanine or cysteine-to-serine mutants for each conserved cysteine

  • Create single and multiple mutations to assess individual and combined effects

• Structural characterization:

  • Circular dichroism to assess secondary structure changes

  • Thermal shift assays to determine effects on stability

  • UV-visible spectroscopy to analyze iron-sulfur cluster incorporation

  • EPR spectroscopy to assess cluster integrity and environment

• Functional assessment:

  • Quantify iron and sulfur content of purified mutants

  • In vitro activity assays with octanoyl-peptide substrate

  • LC-MS analysis to detect reaction intermediates that may accumulate with specific mutations

This systematic approach would provide insights into the structure-function relationships in P. zucineum LipA and could reveal unique features compared to classical LipA enzymes.

What are the kinetic parameters of P. zucineum LipA and how do they compare with other bacterial lipoyl synthases?

Determining and comparing kinetic parameters is essential for understanding the catalytic efficiency of P. zucineum LipA:

Methodology for Kinetic Analysis:

• Steady-state kinetics approach:

  • Vary substrate concentration (octanoyl-peptide): 5-500 μM

  • Measure initial rates at each concentration

  • Plot reaction velocity vs. substrate concentration

  • Determine K_m, k_cat, and k_cat/K_m using appropriate software

• Multiple substrate kinetics:

  • Vary both octanoyl-peptide and SAM concentrations

  • Determine if the reaction follows a sequential or ping-pong mechanism

  • Assess potential cooperativity between substrates

• Pre-steady-state kinetics:

  • Use rapid quench flow or similar techniques to analyze the first turnover

  • Determine rate constants for individual steps in the reaction

  • Identify rate-limiting steps

Comparative Analysis Framework:

This comparative approach would help position P. zucineum LipA within the spectrum of bacterial lipoyl synthases and potentially reveal adaptations specific to its ecological niche.

How does temperature and pH affect the stability and activity of recombinant P. zucineum LipA?

Understanding the environmental parameters that influence LipA activity is crucial for both basic research and optimization of in vitro applications:

Methodological Approach:

• Temperature-activity profile:

  • Conduct activity assays at various temperatures (4°C to 80°C)

  • Measure initial reaction rates at each temperature

  • Plot relative activity versus temperature to determine optimum

• Thermal stability analysis:

  • Incubate enzyme at different temperatures for fixed time periods

  • Measure remaining activity after incubation

  • Calculate half-life at each temperature

  • Perform differential scanning calorimetry (DSC) to determine melting temperature (T_m)

• pH-activity profile:

  • Prepare buffers covering pH range 5.0-10.0 (with consistent ionic strength)

  • Measure enzyme activity at each pH

  • Plot relative activity versus pH to determine optimum

• pH stability profile:

  • Incubate enzyme at different pH values for fixed time periods

  • Adjust pH to optimal value before measuring remaining activity

  • Determine pH range for stability

• Combined effects:

  • Design experiments to test interactions between temperature and pH

  • Generate 3D contour plots of activity as a function of both parameters

Expected Findings and Implications:

Given what is known about lipoyl synthases from different organisms, we might expect:

• Temperature effects:

  • P. zucineum LipA likely has a temperature optimum reflecting its native environment

  • The iron-sulfur clusters may become less stable at higher temperatures, affecting activity

  • Extended incubation at elevated temperatures would likely accelerate cluster degradation

• pH effects:

  • Most lipoyl synthases function optimally at slightly alkaline pH (7.5-8.5)

  • Extreme pH values could affect iron-sulfur cluster stability

  • pH may influence substrate binding and product release

• Buffer components:

  • Certain buffer components (phosphate, certain biological buffers) might interact with iron-sulfur clusters

  • Testing multiple buffer systems at equivalent pH values would be informative

This characterization would provide practical guidelines for handling recombinant P. zucineum LipA and offer insights into its adaptations to its native environment.

How can recombinant P. zucineum LipA be used to study the mechanism of lipoic acid biosynthesis?

Recombinant P. zucineum LipA serves as a valuable tool for elucidating the fundamental mechanisms of lipoic acid biosynthesis:

Research Applications and Methodologies:

• Mechanistic studies using substrate analogs:

  • Synthesize octanoyl-peptides with isotopic labels (¹³C, ²H) at specific positions

  • Track the fate of labeled atoms using mass spectrometry

  • Identify reaction intermediates and their structures

  • Determine the order of sulfur insertion (C-6 vs C-8 position)

• Investigation of reaction intermediates:

  • Use rapid-quench techniques to trap transient intermediates

  • Analyze trapped intermediates by LC-MS

  • Compare with intermediates observed with other LipA enzymes, such as the thiol-octanoyl-peptide observed with T. kodakarensis LipS2

• Study of auxiliary cluster regeneration:

  • Design assays to monitor multiple turnover capability

  • Identify potential carrier proteins from P. zucineum genome

  • Test if NfuA or other carrier proteins can regenerate P. zucineum LipA activity

  • Compare with the established role of NfuA in regenerating LipA from other species

• Comparative analysis with novel lipoyl synthases:

  • Compare the reaction mechanism with the LipS1/LipS2 system from T. kodakarensis

  • Assess if P. zucineum LipA requires partner proteins for full activity

  • Investigate the presence of unique conserved motifs beyond the classical CX₃CX₂C

• Structural studies:

  • Crystallize P. zucineum LipA with substrates or substrate analogs

  • Use cryo-EM to visualize the enzyme in different catalytic states

  • Perform hydrogen-deuterium exchange mass spectrometry to identify conformational changes during catalysis

These approaches would contribute to the broader understanding of lipoic acid biosynthesis across different bacterial species and potentially reveal novel aspects of the reaction mechanism.

What insights can comparative studies of P. zucineum LipA provide about the evolution of lipoic acid biosynthesis pathways?

Comparative analysis of P. zucineum LipA with other lipoyl synthases offers valuable evolutionary insights:

Evolutionary Analysis Framework:

• Phylogenetic positioning:

  • Construct phylogenetic trees including LipA sequences from diverse bacterial species

  • Compare with trees for other lipoic acid metabolism enzymes (LipB, LplA)

  • Identify potential horizontal gene transfer events

  • Position P. zucineum LipA relative to classical LipA and novel LipS1/LipS2 systems

• Structural conservation analysis:

  • Identify conserved and variable regions across LipA homologs

  • Map conservation patterns onto available crystal structures

  • Compare motif architecture between P. zucineum LipA and structurally novel lipoyl synthases like those in T. kodakarensis

• Co-evolution with partner proteins:

  • Analyze genomic context of lipA genes across species

  • Identify potential co-evolution patterns with carrier proteins like NfuA

  • Investigate if patterns exist with other lipoic acid metabolism genes

• Adaptation to ecological niches:

  • Compare LipA enzymes from organisms in different environments

  • Analyze relationships between enzyme properties and habitat

  • Determine if P. zucineum's lifestyle correlates with specific LipA features

Evolutionary Significance:

The discovery of structurally novel lipoyl synthases in archaea suggests that two distinct types of lipoyl synthases have evolved in nature . P. zucineum LipA analysis could provide insights into:

• Whether bacterial lipoyl synthases represent a monophyletic group or if there are multiple evolutionary innovations

• How the regeneration mechanism involving carrier proteins evolved

• Whether the reaction mechanism is strictly conserved or if variations exist across different bacterial lineages

• The evolutionary pressures that shaped the current lipoic acid biosynthesis pathways

This comparative approach would contribute to understanding how this essential metabolic pathway evolved across different domains of life.

How can recombinant P. zucineum LipA be used to investigate disorders related to lipoic acid deficiency?

Recombinant P. zucineum LipA can serve as a valuable research tool for studying human disorders associated with lipoic acid deficiency:

Research Applications in Biomedical Context:

• Model system for human lipoylation defects:

  • Human lipoic acid deficiency syndromes are associated with mutations in lipoic acid synthesis enzymes

  • Bacterial LipA can serve as a simpler model system to understand conserved mechanisms

  • Mutations equivalent to human disease variants can be introduced and studied in the bacterial system

• Development of enzymatic assays:

  • Recombinant P. zucineum LipA could be used to develop assays for:

    • Screening compounds that enhance lipoyl synthase activity

    • Testing small molecules that might bypass lipoylation defects

    • Measuring lipoylation status in biological samples

• Investigation of regeneration mechanisms:

  • Research has shown that humans with defects in iron-sulfur carrier genes have deficiencies of lipoic acid

  • P. zucineum LipA can be used to study how carrier proteins like NfuA maintain enzyme activity

  • These insights could inform therapeutic approaches for human disorders

• Structural insights for drug development:

  • Structural studies of bacterial LipA can inform the development of drugs targeting human lipoylation pathways

  • Understanding the active site and catalytic mechanism could aid in designing molecules that enhance lipoylation

• Enzyme replacement therapy research:

  • Engineered bacterial lipoate protein ligase A (lplA) has been shown to restore lipoylation levels, cellular respiration, and growth in lipoylation null cells

  • Similar approaches could be explored using engineered bacterial LipA to restore lipoic acid synthesis

  • P. zucineum LipA could serve as a platform for engineering enhanced variants

This translational research direction connects basic biochemical studies of bacterial LipA to potential applications in understanding and addressing human metabolic disorders.

What are the potential approaches for engineering P. zucineum LipA for enhanced stability or catalytic efficiency?

Engineering P. zucineum LipA for improved properties presents several promising research avenues:

Protein Engineering Strategies:

• Rational design approaches:

  • Identify residues in the active site pocket through homology modeling

  • Introduce mutations to enhance substrate binding (lower K_m)

  • Modify residues near the iron-sulfur clusters to improve stability

  • Engineer enhanced interfaces with carrier proteins to facilitate cluster regeneration

• Directed evolution:

  • Develop a high-throughput screening assay for LipA activity

  • Generate libraries through error-prone PCR or DNA shuffling

  • Screen for variants with enhanced thermal stability, catalytic activity, or oxygen tolerance

  • Combine beneficial mutations identified in different rounds of selection

• Domain swapping:

  • Exchange domains between LipA enzymes from different species

  • Create chimeric enzymes combining the stability of thermophilic LipA with the activity of mesophilic variants

  • Explore fusion proteins with carrier proteins to enhance cluster regeneration

• Computational design:

  • Use molecular dynamics simulations to identify regions of flexibility

  • Apply computational stability prediction algorithms to suggest stabilizing mutations

  • Model the impact of mutations on cluster coordination and substrate binding

Specific Engineering Targets:

• Oxygen tolerance:

  • Iron-sulfur clusters are oxygen-sensitive, limiting the utility of LipA

  • Engineer variants with improved oxygen tolerance by:

    • Creating more shielded environments for iron-sulfur clusters

    • Introducing sacrificial antioxidant residues

    • Modifying cluster coordination to reduce oxygen reactivity

• Thermal stability:

  • Analyze thermostable LipA variants like those from hyperthermophiles

  • Introduce stabilizing interactions (salt bridges, disulfide bonds) based on these models

  • Rigidify flexible regions while maintaining necessary conformational changes

• Cluster regeneration:

  • Engineer enhanced interaction with carrier proteins like NfuA

  • Create fusion proteins with carrier domains to facilitate in situ cluster regeneration

  • Modify the auxiliary cluster coordination to reduce self-destruction

These approaches could yield LipA variants with enhanced properties for both research applications and potential biotechnological uses.

What aspects of P. zucineum LipA reaction mechanism remain unclear and how might they be addressed?

Despite advances in understanding lipoyl synthase function, several mechanistic aspects remain unresolved:

Outstanding Mechanistic Questions:

• Detailed reaction coordinate:

  • How exactly does the radical SAM chemistry proceed?

  • What is the precise order of events in the two sulfur insertion steps?

  • Are there stable intermediates between the first and second sulfur insertions?

• Cluster regeneration mechanism:

  • How exactly does NfuA or other carrier proteins replace the destroyed auxiliary cluster?

  • What protein-protein interactions facilitate this process?

  • Is regeneration coupled to catalysis or does it occur independently?

• Substrate specificity determinants:

  • What structural features determine which proteins are substrates for lipoylation?

  • How does LipA recognize and position the octanoyl moiety for specific C-6/C-8 modification?

  • Do variations in substrate recognition exist between LipA from different species?

Methodological Approaches to Address These Questions:

• Advanced spectroscopic techniques:

  • Freeze-quench EPR to trap radical intermediates

  • Mössbauer spectroscopy to characterize iron-sulfur cluster states

  • ENDOR or ESEEM to examine the environment of radical species

• Time-resolved structural studies:

  • Time-resolved X-ray crystallography using synchrotron radiation

  • Time-resolved cryo-EM to capture different states of the reaction

  • Single-molecule FRET to observe conformational changes during catalysis

• Novel substrate designs:

  • Stereospecifically deuterated substrates to track hydrogen atom abstraction

  • Fluorinated or methylated octanoyl substrates to trap specific intermediates

  • Photoaffinity labels to capture transient protein-substrate interactions

• Computational approaches:

  • Quantum mechanical/molecular mechanical (QM/MM) simulations of the reaction

  • Free energy calculations for different reaction pathways

  • Molecular dynamics simulations of enzyme-substrate complexes

• Chemical biology approaches:

  • Activity-based protein profiling to identify new interaction partners

  • Crosslinking coupled with mass spectrometry to map protein-protein interfaces

  • In-cell tracking of cluster exchange using genetically encoded fluorescent sensors

Addressing these questions would provide a comprehensive understanding of how LipA functions and could reveal unique aspects of the P. zucineum enzyme compared to other bacterial lipoyl synthases.

How might structural studies of P. zucineum LipA contribute to our understanding of radical SAM enzymes more broadly?

Structural studies of P. zucineum LipA would significantly advance our knowledge of radical SAM enzymes:

Contributions to Radical SAM Enzyme Understanding:

• Mechanistic insights:

  • Lipoyl synthases are unique among radical SAM enzymes in sacrificing their auxiliary iron-sulfur cluster

  • Structural studies could reveal how the enzyme manages this self-destructive chemistry

  • This would provide insights into how radical SAM enzymes control highly reactive radical species

• Auxiliary cluster architecture:

  • The auxiliary cluster coordination in LipA differs from the canonical radical SAM CX₃CX₂C motif

  • Structural characterization would reveal how different cysteine motifs create distinct cluster environments

  • This information could inform understanding of diverse iron-sulfur coordination in other enzymes

• Substrate positioning:

  • How radical SAM enzymes position substrates relative to the deoxyadenosyl radical is a fundamental question

  • Structures with bound substrate would reveal the geometric constraints necessary for hydrogen atom abstraction

  • These principles may apply broadly to other radical SAM enzymes

• Conformational changes:

  • Radical SAM enzymes often undergo conformational changes during catalysis

  • Capturing P. zucineum LipA in different states would provide insights into these dynamics

  • Understanding these movements is crucial for engineering efforts with radical SAM enzymes

Methodological Approaches:

• X-ray crystallography:

  • Crystallize P. zucineum LipA under anaerobic conditions

  • Obtain structures with:

    • Both clusters intact

    • SAM or SAM analogs bound

    • Octanoyl substrate analogs

    • Product analogs

  • Use microcrystal electron diffraction for challenging crystals

• Cryo-electron microscopy:

  • Single-particle cryo-EM analysis of LipA alone and in complex with:

    • Substrates and substrate analogs

    • Carrier proteins like NfuA

    • Other potential protein partners

  • Time-resolved cryo-EM to capture different catalytic states

• Spectroscopic integration:

  • Correlate structural data with spectroscopic findings (EPR, Mössbauer)

  • Combine with computational modeling to understand electronic structures

  • Use structure-guided spectroscopic experiments to probe specific features

These structural studies would provide a blueprint for understanding how radical SAM enzymes harness the extreme reactivity of radical species for controlled chemistry, with implications extending far beyond lipoic acid biosynthesis to the broader family of these important biological catalysts.